The epsilon protein kinase C (epsilonPKC) isozyme has been clearly established as a key mediator of cardiac preconditioning (PC). It is therefore of great interest to study the molecular mechanisms of epsilonPKC-selective signaling to better understand how it mediates protection and to identify potential avenues for the development of epsilonPKC-selective therapeutic modulators of PC. Our current studies indicate that in vitro phosphorylation of an approximately 18 kDa protein found in the particulate cell fraction of cardiac myocytes can be used as a marker of epsilonPKC activation and epsilonPKC-mediated PC. Peptide mass spectrometry and immunoprecipitation analyses have determined that this protein is the IV subunit of cytochrome c oxidase (COIV). Further, this protein is likely an in vivo substrate of epsilonPKC in neonatal cardiac myocytes (NCMs). We hypothesize that COIV plays a key and previously uncharacterized role in cardiac protection against ischemia. Previously we demonstrated that 3 nM 4-beta PMA treatment preferentially activates the epsilonPKC isozyme in NCMs. In this proposal we will determine if epsilonPKC can regulate cytochrome c activity and relate these findings to cardiac PC. We will isolate mitochondria and submitochondrial fractions from NMCs subjected to 3 nM 4-beta PMA treatment and hypoxic PC and monitor cytochrome c oxidase activity, epsilonPKC binding to or phosphorylation of COIV, confocal and electron microscopy COIV and epsilonPKC co-localization studies and mass spectrometric, Cy dye and Pro-Q Diamond methodologies to identify phospho-proteins. Similar analyses will be performed on mitochondria and mitochondrial fractions isolated from adult rat hearts subjected to ischemic PC. The specific aims of this proposal will be: Aim i: To determine the effects of epsilonPKC on cytochrome c oxidase in neonatal cardiac myocytes.; Aim ii To determine the role of epsilonPKC modulation of cytochrome c oxidase in neonatal cardiac myocyte preconditioning and Aim iii: To determine the role of epsilonPKC in the modulation of cytochrome c oxidase in adult rat myocardium exposed to ischemia/reperfusion and ischemic preconditioning. Our work will focus on a novel mechanistic event in the PC paradigm modulated by the epsilonPKC isozyme. A better understanding of the molecular and cellular epsilonPKC targets involved in PC will improve opportunities for the therapeutic application of cardioprotective strategies for patients at risk for myocardial infarction.